Development and evaluation of method to detect rabies virus using nucleic acid sequence based amplification with internal control RNA

Abstract

Standard nucleic acid sequence based amplification (NASBA) method without internal control RNA (IC RNA), although uncertain, it may not avoid false negative. The objective of this study as to develop and to evaluate whether NASBA with internal control RNA (IC-NASBA) could be used for diagnosis of rabies and to avoid false negative without compromising its detection sensitivity when compare to NASBA. In this study, the modified overlap extension by polymerase chain reaction method was used to construct rabies modified fragment containing 25 changed and 42 inserted nucleotides in N gene fragment of rabies virus as internal control in IC-NASBA. Segment of changed nucleotide was a target for specific IC probe. Its longer sequences reduced competitive capacity with WT in amplification reaction. Constructed IC RNA was added to each reaction tube in amplification step. It was amplified by the same target NASBA primers and was detected by a probe complementary to the internal nontarget sequences. IC RNA of 25x10[superscript-4] fg per reaction was used in the amplification step. IC-NASBA was applied in the target of brain and urine specimens. Twenty samples of rabies infected and non-infected brains were examined. The result showed that IC RNA could be detected in all except in high quantity virus samples. Fifteen urine samples of them, previously presence to contain inhibitor, were subject to IC-NASBA assay. Results were in accessed with nested RT-PCR. In conclusion, IC-NASBA should be useful in the diagnosis of rabies and may increase accuracy in rabies detection.